Hematopoiesis is the process by which blood cells develop and differentiate from pluripotent stem cells in the bone marrow. This process involves a complex interplay of polypeptide growth factors (cytokines) acting via membrane-bound receptors on the target cells. cytokine action results in cellular proliferation and differentiation, with response to a particular cytokine often being lineage-specific and/or stage-specific. Development of a single cell type, such as a platelet, from a stem cell may require the coordinated action of a plurality of cytokines acting in the proper sequence.
The known cytokines include the interleukins, such as IL-1, IL-2, IL-3, IL-6, IL-8, etc.; and the colony stimulating factors, such as G-CSF, M-CSF, GM-CSF, erythropoietin (EPO), etc. In general, the interleukins act as mediators of immune and inflammatory responses. The colony stimulating factors stimulate the proliferation of marrow-derived cells, activate mature leukocytes, and otherwise form an integral part of the host's response to inflammatory, infectious, and immunologic challenges.
Various cytokines have been developed as therapeutic agents. For example, erythropoietin, which stimulates the development of erythrocytes, is used in the treatment of anemia arising from renal failure. Several of the colony stimulating factors have been used in conjunction with cancer chemotherapy to speed the recovery of patients' immune systems. Interleukin-2, .alpha.-interferon and .gamma.-interferon are used in the treatment of certain cancers.
An activity that stimulates megakaryocytopoiesis and thrombocytopoiesis has been identified in body fluids of thrombocytopenic animals and is referred to in the literature as "thrombopoietin" (recently reviewed by McDonald, Exp. Hematol. 16:201-205, 1988 and McDonald, Am. J. Ped. Hematol. Oncol. 14:8-21, 1992).
Recently, several groups have identified and/or cloned a protein that binds to the cellular MPL receptor and stimulates megakaryocytopoiesis and thrombocytopoiesis. See, de Sauvage et al., Nature 369:533-538, 1994; Lok et al., Nature 369:565-568, 1994; Kaushansky et al., Nature 369:568-571, 1994; Wendling et al., Nature 369:571-574, 1994; and Bartley et al., Cell 77:1117-1124, 1994. It has been proposed that this protein be termed thrombopoietin (Kaushansky et al., ibid.). Although this protein has been shown to stimulate platelet production in vivo (Kaushansky et al., ibid.), it appears to be subject to proteolysis and was isolated in heterogeneous or degraded form (Bartley et al., ibid.; de Sauvage et al., ibid.).
Proteolysis and heterogeneity are significant problems that can impede the development of new pharmaceutical agents. There remains a need in the art for homogeneous, undegraded preparations of thrombopoietin and for methods of making such preparations. The present invention fulfills these needs and provides other, related advantages.